Rotator cuff and biceps tendons that appeared grossly normal were procured from adult cadavers without a history of shoulder problems. These tendons were analyzed for the amount and type of glycosaminoglycan, type of proteoglycan, and histology. When compared with the distal/tensional region of biceps tendon, the glycosaminoglycan content of supraspinatus, infraspinatus, and subscapularis tendons was 2.5-fold higher and the glycosaminoglycan content of the proximal/compressed region of biceps tendon was 3-fold higher. The ratio of hyaluronic acid to chondroitin sulfate/dermatan sulfate in all three cuff tendons was approximately 1. Rotator cuff tendons contained large proteoglycan similar to aggrecan, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel migration, elution from Sepharose CL-4B, and content of both chondroitin sulfate and keratan sulfate chains. Both decorin and biglycan were also present, as demonstrated by migration in sodium dodecyl sulfate-polyacrylamide gels and core protein immunoreactivity. In contrast, decorin was the only proteoglycan prominent in distal/tensional regions of biceps tendon. Histological analysis showed layers of loosely organized, alcian blue-stained material running between the longitudinal collagen fiber bundles. The proteoglycan content of rotator cuff tendons was similar to fibrocartilage in tendons that have been subjected to compressive loads in situ. This suggests that cells of normal adult rotator cuff tendons have adapted to loads distinct from pure tension. However, the histological organization did not resemble mature fibrocartilage. The increased amount of proteoglycan in rotator cuff tendons may serve to separate and lubricate collagen bundles as they move relative to each other during normal shoulder motion.